Abstract The tongue is a heterogeneous tissue that comprises taste fungiform papillae (FP) and non-taste filiform papillae (FILIF) in the anterior region. Maintenance of FP and FILIF requires tight regulation of basal epithelial cell renewal and subsequent proper differentiation. The basal epithelia of FP and FILIF differentiate into distinct sub-sites of the tongue, suggesting significant differences in gene regulation. Understanding the genes that regulate the maintenance of these distinct taste and non-taste epithelia will provide fundamental insight into the mechanisms that control tongue function. Our data establish that Hedgehog (HH) signaling in epithelial cells is essential for adult FP maintenance. Strikingly, within the epithelium, expression of the HH transcriptional effector and target gene, Gli1, is restricted to the FP, while the related transcription factor and key HH pathway component, Gli2 is expressed in the entire basal lingual epithelium, including FP and FILIF. Although we have demonstrated an essential role for epithelial Gli2 in FP maintenance, there remains a major gap in understanding how GLI2 regulates FP homeostasis. Further, while essential for many oral functions, non-taste FILIF homeostasis remains understudied. We hypothesize that FP and FILIF epithelia have distinct gene profiles that differentially regulate lingual tissue homeostasis. We will use Gli1 reporters to efficiently distinguish between taste and non-taste epithelia. In Aim 1 we will discover differentially expressed genes and their regulation by HH signaling in the FP and FILIF epithelial cell populations employing RNA-seq simultaneously with GLI2 ChIP-seq, using a novel FLAG-tagged knock-in Gli2 allele. Our published data show that inhibition of HH signaling using the cancer drug sonidegib is sufficient to drive taste organ and sensation loss whereas the non-taste FILIF remain intact. Notably, patients who take sonidegib report severe taste dysgeusia and ageusia. Building on studies to understand the mechanisms of taste alteration after pharmacological inhibition of HH pathway, we will determine deregulated genes and their function. Although expression of Gli1 is eliminated after sonidegib treatment, our preliminary data indicate that Gli2 expression is retained in the FP epithelium. In Aim 2, to understand the exact effects of HH pathway blockade and role of active Gli2 in FP and FILIF, we will rely on similar experimental approaches combining RNA-seq and GLI2 ChIP-seq. The data obtained will help to understand and illustrate mechanisms that underlie taste disruptions in patients receiving HH pathway inhibitor drugs and may lead to identification of novel targets for development of therapeutic approaches. Principally, defining gene expression and regulation in the distinct taste (Gli1+;Gli2+) and non-taste (Gli1-;Gli2+) epithelia will confer advanced knowledge about the mechanisms of tongue homeostasis. Overall, this proposal will lay the foundation for an in-depth understanding of the genetic, molecular and functional architecture of the FP and FILIF basal epithelium and HH signaling regulation in these particular taste and non-taste lingual environments.